Establishing spatial organization is a fundamental requirement for all living systems. A critical element in cellular organization is cell polarity - the presence of a directed axis that controls processes such as structural assembly, organelle transport, and localization of macromolecules. Cell polarity is required for nearly all cell types and for diverse physiological processes, such as cell differentiation, cell motility, neuronal growth, and immune responses. In the life cycle of the budding yeast Saccharomyces cerevisiae, cell polarity is critical for mating and bud formation. The Rho-family GTPase Cdc42 is a key regulator and readout of cell polarization and accumulation of activeCdc42 at specific sites on the plasma membrane is required for polarization. Polarization normally occurs in response to spatial and temporal cues. However, in the absence of asymmetric inductive signals, yeast cells still have the ability to spontaneously polarize, albeit in random directions. We hypothesize that cell polarity is brought about through a coupling of two processes: recognition of asymmetric cues, and activation of an intrinsic, spontaneous mechanism to break symmetry. The overarching aim of this investigation is to dissect, disentangle, and reassemble mechanisms of polarization in the yeast budding pathway. Our approach takes advantage of the combined strength of mathematical modeling and molecular genetics in yeast. The proposed research has three goals: The first two investigate two mechanisms of spontaneous polarization of Cdc42, one dependent and the other independent of the cytoskeleton. The third aim examines how localization signals, amplified through a GTPase cascade, bias these spontaneous mechanisms of polarization to yield a single, robust, specified site of Cdc42 polarization.